Cells and tissues can become deficient in particular biomolecules due to, for example, stressful environmental conditions. In order for the cells and tissues to survive, the levels of deficient biomolecules must be raised within the cells and tissues to meet metabolic demand. For example, ATP is a biomolecule relied upon by cells as a primary source of energy and an increased metabolic demand or shortfall in supply of ATP to cells can result in death of the cells if demand is not met quickly.
ATP is the fuel that powers all cells-animal, plants, bacteria, fungi, etc. Such as a car without gas, humans and other creatures with an empty ATP “tank” do not go. In fact, they die. The energy derived from the breakdown of nutrients is ultimately conserved in the high energy phosphate bonds of ATP. When these bonds are broken, they provide accessible energy to cells, tissues, organs and organ systems. Cells constantly synthesize and metabolize ATP. ATP can be produced either aerobically through oxidative phosphorylation, with oxygen as the terminal electron acceptor and yielding carbon dioxide (CO2) and water as by-products, or anaerobically during glycolysis. While glycolysis can provide energy to cells, the supply is limited because the cellular environment becomes acidic, injuring the cell and inhibiting ATP production.
The vascular circulatory system delivers a continuous supply of energy that is derived from oxygen and nutrients. In the vasculature, a barrier of endothelial cells separates the cells being fed from the vessel lumen. To reach cells outside of the vasculature, oxygen and nutrients must pass through the endothelial lining into the interstitial space. The flow of blood, and thus the flow of nutrients and oxygen can be cut off or reduced as a result of disease or trauma. For example, myocardial infarction (heart attack), stroke, hypotension and severe trauma, such as severing a carotid artery in an automobile accident, result in loss of oxygen, leading to the loss of homeostasis, and possibly resulting in death.
When blood supply is re-established after an ischemic event, an event that results in the loss of oxygen and nutrients to tissue, ischemia-reperfusion injury can occur. As the cells attempt to synthesize ATP, after reoxygenation, toxic metabolites are produced, such as free radicals. Ischemia is not only an injury- or disease-related phenomenon, but can be induced as a side effect of surgeries, such as aortic bypass, open heart surgery, major tissue reconstruction, tumor removal, intestinal resection and organ transplantation.
Ischemia represents an enormous challenge to successful tissue and organ transplantation. About 14,000 kidneys and 2500 hearts are transplanted in the United States each year. After removal, organs have a limited life span in the absence of nutrients and oxygen. Hearts must be transplanted within 4 to 6 hours after harvest, while kidneys must be transplanted within 72 hours. Because recipients are often far from donors, these short viability times hamper transplantation. Blood can be stored for about only 45 days at 4° C. and then must be discarded. More complicated is the acquisition of autologous blood in anticipation of surgery. Patients can usually only provide two units of blood in the 45 days. This amount does not suffice, because many surgical procedures use three, four or more units of blood.
Several attempts have been made to overcome or inhibit the detrimental effects of low oxygen supplies. These approaches include: (1) providing glycolytic intermediates to augment anaerobic ATP production; (2) reducing metabolic demand, such as storing cells, tissues and organs at 4° C.; and (3) adding ATP directly to the cells, tissues or organs. Supplying energy to cells would be preferably accomplished by direct administration of ATP; however, cells take up exogenous ATP poorly because they lack ATP receptors or channels. Furthermore, cell plasma membranes are hydrophobic, while ATP is hydrophilic, preventing the ATP from passing through. Introducing ATP into the blood stream is ineffective because ATP cannot cross the endothelial barrier, and ATP is prone to hydrolysis. In addition, ATP is a purinergic receptor agonist and when administered intravenously, ATP can result in vasodilation and hypotension. Attempts to use liposomes to deliver ATP have been largely unsuccessful and inefficient (Arakawa et al. 1998, Puisieux et al. 1994). For example, Puisieux et al. constructed phosphatidyl choline, cholesterol and phosphatidyl serine lipid vesicles that encapsulated ATP, then incubated the vesicles with sperm cells, liver and brain tissue. Although some uptake was observed, controlled delivery matching metabolic demand for ATP was not achieved. When administered in the blood stream, liposomes are usually unable to breach the endothelial cell barrier; in addition, they usually do not have high rates of fusion with cellular membranes, a necessary event for the vesicle to deliver its ATP payload into the cells.
Animal cell plasma membranes contain four major phospholipids that represent greater than half of the total lipid:phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and sphingomyelin. Phosphatidylcholine and sphingomyelin are found mostly in the outer leaflet, while phosphatidylethanolamine and phosphatidylserine are found principally in the inner leaflet. Phosphatidylcholine is the most abundant phospholipids found in animal cells. Thus, any liposomal delivery system should be composed primarily of this phospholipids. Furthermore, phosphatidylcholine is the only naturally occurring phospholipids that forms closed lipid vesicles, which protects the intravesicular contents and reduces leakage. Plasma membranes help maintain cellular integrity and are selectively permeable. While some molecules are able to diffuse through membranes, most, including ATP, require other means to enter, such as transport proteins or channels.
Therefore, there continues to be a need for new approaches to deliver biomolecules to cells for a variety of applications, including but not limited to providing biomolecules, such as ATP, to cells and tissues not receiving sufficient quantities of the biomolecules to meet metabolic demand.